Hydrogen-powered road vehicles : the health benfits and drawbacks of a new fuel

Because of the political, social and environmental problems associated with dependency on fossil fuels, there is considerable interest in alternative energy sources. Hydrogen is regarded as a promising option, particularly as a fuel for road vehicles. The Dutch Energy Research Centre (ECN) recently published a vision of the future, in which it suggested that by 2050 more than half of all cars in the Netherlands could be running on hydrogen. A switch to using hydrogen as the primary energy source for road vehicles would have far-reaching social consequences. As with all technological developments, opportunities would be created, but drawbacks would inevitably be encountered as well. Some of the disadvantages associated with hydrogen are already known, and are to some degree manageable. It is likely, however, that other drawbacks would come to light only once hydrogen-powered cars were actually in use. With that thought in mind, and in view of the social significance of a possible transition to hydrogen, it was decided that the Health Council should assess the positive and negative effects that hydrogen use could have on public health. It is particularly important to make such an assessment at the present early stage in the development of hydrogen technologies, so that gaps in existing scientific knowledge may be identified and appropriate strategies may be developed for addressing such gaps. This report has been produced by the Health and Environment Surveillance Committee, which has special responsibility for the identification of important correlations between environmental factors and public health

Zebrafish as a model to study the role of Peroxisome Proliferating-Activated Receptors in adipogenesis and obesity

The Peroxisome Proliferator-Activated Receptors (PPARs) PPARA and PPARD are regulators of lipid metabolism with important roles in energy release through lipid breakdown, while PPARG plays a key role in lipid storage and adipogenesis. The aim of this review is to describe the role of PPARs in lipid metabolism, adipogenesis, and obesity and evaluate the zebrafish as an emerging vertebrate model to study the function of PPARs. Zebrafish are an appropriate model to study human diseases, including obesity and related metabolic diseases, as pathways important for adipogenesis and lipid metabolism which are conserved between mammals and fish. This review synthesizes knowledge on the role of PPARs in zebrafish and focuses on the putative function of PPARs in zebrafish adipogenesis. Using in silico analysis, we confirm the presence of five PPARs (pparaa, pparab, pparda, ppardb, and pparg) in the zebrafish genome with 67–74% identity to human and mouse PPARs. During development, pparda/b paralogs and pparg show mRNA expression around the swim bladder and pancreas, the region where adipocytes first develop, whereas pparg is detectable in adipocytes at 15 days post fertilization (dpf). This review indicates that the zebrafish is a promising model to investigate the specific functions of PPARs in adipogenesis and obesity.This research is financially supported by Netherlands Organisation for Scientific Research (NWO) VIDI/864.09.005 and ASPASIA/015.006.018

Shifting CCR7 towards Its Monomeric Form Augments CCL19 Binding and Uptake.

The chemokine receptor CCR7, together with its ligands, is responsible for the migration and positioning of adaptive immune cells, and hence critical for launching adaptive immune responses. CCR7 is also induced on certain cancer cells and contributes to metastasis formation. Thus, CCR7 expression and signalling must be tightly regulated for proper function. CCR7, like many other members of the G-protein coupled receptor superfamily, can form homodimers and oligomers. Notably, danger signals associated with pathogen encounter promote oligomerisation of CCR7 and is considered as one layer of regulating its function. Here, we assessed the dimerisation of human CCR7 and several single point mutations using split-luciferase complementation assays. We demonstrate that dimerisation-defective CCR7 mutants can be transported to the cell surface and elicit normal chemokine-driven G-protein activation. By contrast, we discovered that CCR7 mutants whose expression are shifted towards monomers significantly augment their capacities to bind and internalise fluorescently labelled CCL19. Modeling of the receptor suggests that dimerisation-defective CCR7 mutants render the extracellular loops more flexible and less structured, such that the chemokine recognition site located in the binding pocket might become more accessible to its ligand. Overall, we provide new insights into how the dimerisation state of CCR7 affects CCL19 binding and receptor trafficking

Development and application of in vitro and in vivo reporter gene assays for the assessment of (xeno-)estrogenic compounds in the aquatic environment

In recent years, both scientific and public concern about the possible threat of estrogenic compounds in the environment that may impact the reproduction of humans and wildlife has increased. Many substances have been demonstrated to possess estrogenic potency using in vitro test systems, and these compounds have been identified in the environment using chemical analysis. However, up until now, it has been difficult to rapidly estimate the total biologically active estrogenic potency in environmental mixtures, as well as predict the exposure of and possible effects on organisms. The research presented in this thesis describes the development and application of two new bioassays to determine the biologically active exposure of natural and xenobiotic estrogens (xeno-estrogens) in the environment and predict the estrogenic effects of such exposure on fish. These new bioassays may help to determine if organisms are exposed to levels of (xeno-)estrogens which may impact their reproduction or endocrine functioning.An in vitro estrogen receptor (ER)-mediated chemical activated luciferase reporter gene expression (ER-CALUX) assay was developed by stable transfection of an ER regulated luciferase reporter gene in the T47D human breast cancer cell line. The ER-CALUX is very sensitive and highly responsive to natural estrogens, such as estradiol (E2), with a detection limit of 0.5 pM E2, an EC50 of 6 pM E2 and a maximum induction of 100-fold at 30 pM relative to solvent controls. The presence of anti-estrogenic activity can be determined by co-incubation with E2, and was demonstrated for ICI 182,780, TCDD and tamoxifen. A variety of xeno-estrogenic compounds are estrogenic in the ER-CALUX assay, including pesticides, alkylphenols, phthalates and phytoestrogens, though with a potency 10,000 to 100,000 times less than E2. In comparison to a widely used recombinant yeast screen, the ER-CALUX assay was more sensitive to both (xeno-)estrogens and anti-estrogens. Given the exquisite sensitivity of the ER-CALUX assay, it can be an ideal screening tool for determining estrogenic activity in complex mixtures from various environmental matrices, such as water, sediment and particulate matter. A number of sediment extracts obtained from industrialized areas such as the Port of Rotterdam showed high estrogenic potency. In addition, a simple method was developed to determine if estrogen metabolites present in human urine and fish bile could be reactivated into active estrogens. Estrogen glucuronides in urine obtained from human males and females were deconjugated by enzymatic hydrolysis following incubation with ß-glucuronidase or live E. coli cells, indicating that bacterial hydrolysis as present in wastewater treatment plants may form a secondary source of estrogenic substances in the environment. Also, glucuronides in bile samples from male fish showed estrogenic activity following deconjugation. These conjugated metabolites could also form a secondary source of estrogen exposure in fish due to enterohepatic recycling. High estrogenic activity of deconjugated bile metabolites from male bream correlated with elevated estrogenic potency in water extracts as well as vitellogenin induction in blood of the same fish.An in vivo reporter gene assay using transgenic zebrafish was developed by stably introducing an estrogen binding sequence linked to a TATA box and luciferase reporter gene into the genome. This is the first report of an in vivo reporter gene assay to assess exposure to estrogen modulating substances. Exposure of the transgenic zebrafish to E2 during juvenile stages revealed the period of gonad differentiation to be the most responsive early life stage. In this juvenile period, E2 induced luciferase activity in a dose-dependent manner. Also the natural estrogens estrone (E1) and 17α-estradiol, as well as the synthetic estrogens diethystilbestrol and ethinylestradiol (EE2), and the organochlorine pesticide o,p'DDT demonstrated estrogenic activity in vivo in this test system. In adult male transgenic zebrafish, short-term exposure to E2 revealed a number of tissues such as liver, bone, muscle and scales to be a potential target organ for E2. However, the testis was the most sensitive and responsive target tissue to estrogens. Exposure to E2 resulted in dose-response related luciferase induction in the testis. Immunohistochemistry revealed the Sertoli and germ cells to be the main target cells of E2 in the zebrafish testis. ERαandβmRNA was detected already in young embryos (24 hours post fertilization) and both subtypes were highly expressed during the period of gonad differentiation. In adult male transgenics, tissues with the highest luciferase activity (testis and liver) expressed both ERαandβmRNA.Exposure of ER-CALUX cells is very direct without interaction with environmental factors influencing bioavailability and accumulation and without the toxicokinetics of an intact organism. In the transgenic fish assay, however, these factors do play a role, resulting in concentrations in the cell which are sometimes difficult to predict using an in vitro system. Both assays exhibited high sensitivity to natural and synthetic estrogens such as E2, E1 and EE2, though differences in their relative potencies to E2 were found. EE2 was 100 times more potent than E2 in the transgenic fish, whereas it was only slightly (1.2 times) more potent than E2 in the ER-CALUX assay. The highly accumulating xeno-estrogen o,p'DDT was estrogenic in both assays, with similar potencies relative to E2. On the other hand, xeno-estrogenic chemicals with which are readily metabolized in vivo such as nonylphenol and bisphenol A were estrogenic in vitro but did not induce reporter gene expression in vivo in short- term exposures. Estrogenic activity was also found in mixtures reflecting concentrations of (xeno-)estrogens found in effluents of a domestic wastewater treatment plant, using both the ER-CALUX and transgenic zebrafish assays. In the transgenic zebrafish, levels of estrogenic activity found in this effluent exceed levels reported in the literature necessary to induce vitellogenin induction and inhibit testicular growth and reproduction in other fish species. This indicates that fish at this particular location may be exposed to (xeno-)estrogens at levels that may impact their reproductive success.This study resulted in the development of two important new biological tools to identify estrogenic compounds. These tools are very valuable in determining if substances in the environment are present at levels that may interfere with reproduction or endocrine functioning in organisms. The in vitro ER-CALUX assay with T47D cells is the most sensitive and responsive stably transfected reporter gene assay reported to date and has already proven indispensable in rapidly and cost-effectively determining total biological potency of estrogenic chemicals in complex environmental mixtures. The in vivo transgenic zebrafish assay is the ideal complement to the ER-CALUX assay because environmental chemistry and toxicokinetics of compounds are taken into account. This assay is important for predicting the estrogenic effects of chemicals on fish. The surprising result that the male testis, and not the liver, is the most sensitive and responsive target tissue to estrogens underlines the vulnerability of the male reproductive organs and provides a scientific basis for the world-wide observance of male gonad abnormalities in wild populations of fish. These newly developed techniques have demonstrated that though probably not widespread, there are some localized areas of concern of elevated (xeno-)estrogenic exposure in the Dutch aquatic environment.</p

Distinct Fates of Chemokine and Surrogate Molecule Gradients: Consequences for CCR7-Guided Dendritic Cell Migration.

Chemokine-guided leukocyte migration is a hallmark of the immune system to cope with invading pathogens. Intruder confronted dendritic cells (DCs) induce the expression of the chemokine receptor CCR7, which enables them to sense and migrate along chemokine gradients to home to draining lymph nodes, where they launch an adaptive immune response. Chemokine-mediated DC migration is recapitulated and intensively studied in 3D matrix migration chambers. A major caveat in the field is that chemokine gradient formation and maintenance in such 3D environments is generally not assessed. Instead, fluorescent probes, mostly labelled dextran, are used as surrogate molecules, thereby neglecting important electrochemical properties of the chemokines. Here, we used site-specifically, fluorescently labelled CCL19 and CCL21 to study the establishment and shape of the chemokine gradients over time in the 3D collagen matrix. We demonstrate that CCL19 and particularly CCL21 establish stable, but short-distance spanning gradients with an exponential decay-like shape. By contrast, dextran with its neutral surface charge forms a nearly linear gradient across the entire matrix. We show that the charged C-terminal tail of CCL21, known to interact with extracellular matrix proteins, is determinant for shaping the chemokine gradient. Importantly, DCs sense differences in the shape of CCL19 and CCL21 gradients, resulting in distinct spatial migratory responses

First year growth in relation to prenatal exposure to endocrine disruptors - A dutch prospective cohort study

Growth in the first year of life may already be predictive of obesity later in childhood. The objective was to assess the association between prenatal exposure to various endocrine disrupting chemicals (EDCs) and child growth during the first year. Dichlorodiphenyldichloroethylene (DDE), mono(2-ethyl-5-carboxypentyl)phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono(2-ethyl-5-oxohexyl)phthalate (MEOHP), polychlorinated biphenyl-153, perfluorooctanesulfonic acid, and perfluorooctanoic acid were measured in cord plasma or breast milk. Data on weight, length, and head circumference (HC) until 11 months after birth was obtained from 89 mother-child pairs. Mixed models were composed for each health outcome and exposure in quartiles. For MEOHP, boys in quartile 1 had a higher BMI than higher exposed boys (p = 0.029). High DDE exposure was associated with low BMI over time in boys (0.8 kg/m2 difference at 11 m). Boys with high MECPP exposure had a greater HC (1.0 cm difference at 11 m) than other boys (p = 0.047), as did girls in the second quartile of MEHHP (p = 0.018) and DDE (p < 0.001) exposure. In conclusion, exposure to phthalates and DDE was associated with BMI as well as with HC during the first year after birth. These results should be interpreted with caution though, due to the limited sample size

Prenatal exposure to endocrine disrupting chemicals in relation to thyroid hormone levels in infants – a Dutch prospective cohort study

Background: Endocrine disrupting chemicals (EDCs) present in the environment may disrupt thyroid hormones, which in early life are essential for brain development. Observational studies regarding this topic are still limited, however as the presence of chemicals in the environment is ubiquitous, further research is warranted. The objective of the current study was to assess the association between exposure markers of various EDCs and thyroxine (T4) levels in newborns in a mother-child cohort in the Netherlands. Methods: Exposure to dichlorodiphenyldichloroethylene (DDE), three di-2-ethylhexyl phthalate (DEHP) metabolites, hexachlorobenzene (HCB), polychlorinated biphenyl (PCB)-153, perfluorooctanesulfonic acid (PFOS), and perfluorooctanoic acid (PFOA) was determined in cord plasma or breast milk, and information on T4 levels in heel prick blood spots was obtained through the neonatal screening programme in the Netherlands. Linear regression models were composed to determine associations between each of the compounds and T4, which were stratified for gender and adjusted for a priori defined covariates. Results: Mean T4 level was 86.9 nmol/L (n = 83). Girls in the highest quartile of DDE and PFOA exposure showed an increased T4 level compared to the lowest quartile with both crude and fully adjusted models (DDE > 107.50 ng/L, +24.8 nmol/L, 95% CI 0.79, 48.75; PFOA > 1200 ng/L, +38.6 nmol/L, 95% CI 13.34, 63.83). In boys a lower T4 level was seen in the second quartile of exposure for both PFOS and PFOA, however after fully adjusting the models these associations were attenuated. No effects were observed for the other compounds. Conclusion: DDE and perfluorinated alkyl acids may be associated with T4 in a sex-specific manner. These results should however be interpreted with caution, due to the relatively small study population. More research is warranted, as studies on the role of environmental contaminants in this area are still limited

Transcriptional and epigenetic mechanisms underlying enhanced in vitro adipocyte differentiation by the brominated flame retardant BDE-47

Recent studies suggest that exposure to endocrine-disrupting compounds (EDCs) may play a role in the development of obesity. EDCs such as the flame retardant 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47) have been shown to enhance adipocyte differentiation in the murine 3T3-L1 model. The mechanisms by which EDCs direct preadipocytes to form adipocytes are poorly understood. Here, we examined transcriptional and epigenetic mechanisms underlying the induction of in vitro adipocyte differentiation by BDE-47. Quantitative high content microscopy revealed concentration-dependent enhanced adipocyte differentiation following exposure to BDE-47 or the antidiabetic drug troglitazone (TROG). BDE-47 modestly activated the key adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) in COS7 cells, transiently transfected with a GAL4 reporter construct. Increased gene expression was observed for Pparγ2, leptin (Lep), and glucose-6-phophatase catalytic subunit (G6pc) in differentiated 3T3-L1 cells after BDE-47 exposure compared to TROG. Methylation-sensitive high resolution melting (MS-HRM) revealed significant demethylation of three CpG sites in the Pparγ2 promoter after exposure to both BDE-47 and TROG in differentiated 3T3-L1 cells. This study shows the potential of BDE-47 to induce adipocyte differentiation through various mechanisms that include Pparγ2 gene induction and promoter demethylation accompanied by activation of PPARγ, and possible disruption of glucose homeostasis and IGF1 signaling